ABSTRACT
In this paper we describe the preparation of a series of new phosphorescent labelling reagents, based on monosubstituted palladium(II) coproporphyrin-I and the isothiocyanato reactive group. The labelling reagents differ with respect to the chemical composition of the linker unit that combines the reactive group and the porphyrin chromophore. Altogether, seven different labelling reagents are prepared. The new labelling reagents are conjugated with monoclonal mouse IgG to yield label conjugates with variable degrees of conjugation. The effect is studied of linker unit on: (a) the conjugation reaction kinetics; (b) the biological activity of the resulting IgG conjugates; and (c) the efficiency of phosphorescence emission. The results show that an increase in the length of the linker unit has a positive effect on both the reactivity of the label and the biological activity of the resulting conjugates. In addition, the results indicate that the labels with the most hydrophilic linker units exhibit the highest phosphorescence emission efficiencies.
Subject(s)
Coproporphyrins/chemistry , Immunoconjugates/chemistry , Indicators and Reagents/chemical synthesis , Luminescence , Palladium/chemistry , Animals , Antibodies, Monoclonal/chemistry , Coproporphyrins/chemical synthesis , Cross-Linking Reagents/chemistry , Fluorometry/methods , Immunoglobulin G/chemistry , Isothiocyanates/chemistry , Mice , Models, Chemical , Molecular Structure , Staining and LabelingABSTRACT
A sensitive, specific method for the measurement of uroporphyrinogen decarboxylase(porphyrinogen carboxy-lyase, EC4.1.1.37) in liver homogenates, haemolysates and other tissues is described. Pentacarboxylic porphyrinogen III, prepared from the corresponding porphyrin by reduction with sodium-mercury amalgam, is used as substrate. After oxidation and methyl esterification the reaction product, coproporphyrinogen III, is separated from the substrate and measured by high-performance liquid chromatography, with mesoporphyrin IX as internal standard. The method can readily be adapted for use with any of the other substrates of uroporphyrinogen decarboxylase, including uroporphyrinogens I and III.
Subject(s)
Carboxy-Lyases/analysis , Porphyrinogens/metabolism , Uroporphyrinogen Decarboxylase/analysis , Animals , Chemical Phenomena , Chemistry, Physical , Coproporphyrins/chemical synthesis , Female , Hydrogen-Ion Concentration , Liver/metabolism , Oxidation-Reduction , Porphyrinogens/chemical synthesis , Porphyrins/chemical synthesis , Rats , Rats, Inbred StrainsABSTRACT
N-Methyl mesoporphyrin was a powerful inhibitor of protohaem ferro-lyase in vitro, whereas N-ethyl mesoporphyrin and N-methyl coproporphyrin were not and neither was the newly described green pigment produced by giving rats ethylene. This suggests that the size of the substituent at a pyrrole nitrogen and also the number of carboxylic acid side chains of the substituted porphyrin are important for the inhibitory effect. Evidence that N-methyl mesoporphyrin inhibited the enzyme, whereas the ethylene-derived pigment did not, was also obtained in vivo.